Wider Stoke's shifts, greater water solubility and photostability are key requirements for general acceptance of any new fluorophore. Narrow Stoke's shifts make it difficult to distinguish between actual signal and background signal, and when short wavelength excitation and emission are used, as in dark field fluorescence microscopy, the strong background fluorescence from biological samples can interfere heavily. Since most biological experiments are performed in aqueous solution, fluorescent probes with good water solubility are desirable. Finally, the desired improvements in sensitivity, which drives all attempts to develop new fluorescent labels, requires fluorophores that are photostable and have high quantum yield will be able to achieve high sensitivity.
The potential of pyrene and its derivatives for use in developing improved permanently fluorescent labels for the detection of biological molecules in research and diagnostic applications including fluorescence microscopy, gel electrophoresis, flow cytometry, immunoassays, DNA sequencing, immuno-blotting, nucleic acid probe assays has been under study for more than a century. As a general rule, however, the compounds that were developed proved unsatisfactory because they suffered from narrow Stoke's shifts, photo-instability, low quantum yields, short wave excitation and emissions and/or poor water solubility.
The literature on pyrene and its derivatives regarding uses for fluorescence detection is vast and extends back to the dye chemists of Bayer at the turn of the past century, (cf. Tietze, E., and Bayer, O., Pyrene Sulfoacids and its Derivatives, German Reich Patents 343147, 659883, 233934, 664652, and 658780), although the focus of much of this work has been largely on the development of fluorogenic substrates for use in measuring enzymatic activities. Beginning in the 1970s, numerous labs attempted to develop fluorescent labels based on pyrene, with special emphasis on simple derivatives that could be covalently attached though monofunctional substituents on the polyaromatic rings of pyrene itself. Early examples included: (1) synthesis of N-(3-pyrene maleimide) which was subsequently conjugated with proteins and used in early studies of fluorescence polarization (Weltman, J. K, et al, N-(3-pyrene) maleimide: a Long Lifetime Fluorescent Sulfhydryl Reagent J. Biol. Chem. 248(9), 3173-3177 (1973)). The same maleimide derivative found continued use through the 1990s in a variety of different biomolecular targets including: (i) phosphatases (Csortos, C., et al, Interaction of the Catalytic Subunits of Protein Phosphatase-1 and 2-A with Inhibitor-1 and 2: A fluorescent Study with Sulfhydryl specific Pyrene Maleimide BBRC 169 (2), 559-564 (1990)), and, (ii) of the interaction of the fluorophore with nucleoside analogs (Karim, A. S., et al, Maleimide-mediated protein conjugates of nucleoside triphosphate gamma-S and an internucleotide phosphorothiate diester, Nucleic Acids Research, 23 (11), 2037-2040 (1995)). These compounds suffered from two major limitations, however: (i) poor water solubility of the core pyrene molecule, and, (ii) fluorescence quenching in aqueous buffers.
In an effort to overcome the solubility limitations, numerous labs began to experiment with 8-aminonaphthalene-1,3,6-trisulphonic (“ANTS”) acid derivatives of pyrene, particularly as a label for use in identifying glycoconjugates in electrophoretic gels (cf., (i) Jackson, P., The use of polyacrylamide-gel electrophoresis for the high resolution separation of reducing saccharides labelled with the fluorophore 8-aminonaphtalene-1,3,6-trisulphonic acid, Biochem. J. 270, 705-713 (1990), and, (ii) Chiesa, C., and O'Neill, R. A., Capillary zone electrophoresis of oligosaccharides derivatized with various aminonaphthalene sulfonic acids, Electrophoresis 15, 1132-1140 (1994), (iii) Evangelista, R. A., Guttman, A., and Chen, Fu-Tai, Acid-catalyzed reductive amination of aldoses with 8-amino-pyrene-1,3,6-trisulfonate, Electrophoresis 17, 347-351 (1996), Guttman, A. and Pritchett, T., Capillary gel electrophoresis separation of high-mannose type oligosaccharides derivatized by 1-aminopyrene-1,6,8-trisulfonic acid, Electrophoresis 16, 1906-1911 (1995), and, (iv) Evangelista, R. and Chen, Fu-tai, Analysis of mono-and oligosaccharide isomers derivatized with 9-aminopyrene-1,4,6-trisulfonate by capillary electrophoresis with laser-induced Fluorescence, Analytical Biochemistry 230, 273-280 (1995)). A related derivative, 5-(2-(iodoacetyl)-amino) ethyl) aminonaphtalene-1-sulfonic acid (“1,5-I-AEDANS”) was used in functionalizing nucleosides (cf., Agrawal, S, and Zamecnik, P. C. Site specific functionalization of oligonucleotides for attaching two different reporter groups, Nucleic Acids Research 18 (18), 5419-5423 (1996)). In another application of pyrene to nucleoside labeling, Crisp and Gore reported (Crisp, G. T. and Gore, J., Palladium-catalysed Attachment of Labels with Acetylenic Linker Arms to Biological Molecules, Tetrahedron 53 (4), 1523-1544 (1997)) coupling of the core pyrene fluorophore through propoargylglycine spacers to the 8-alynyl derivatives of adenosine and guanosine, however, this work was never applied to labeling of any biomolecule owing to the quenching limitations noted earlier for the core pyrene fluorophore in aqueous solvents.
Quite different types of applications were developed by Nomura et al and, separately, Wolfbeis et al. In the first, 8-hydroxy-1,3,6-pyrenetrisulfonate has been conjugated to lipids to make the fluorophore more hydrophobic and the conjugates used to measure energy transfer in surfactant vesicles (Nomura, T., et al, Aspects of Artificial Photosynthesis. Energy Transfer in Cationic Surfactant Vesicles, JACS 102 (5), 1484-1488 (1980)). Koller and Wolfbeis extended the much earlier work of Tietze et al, throughout the 1980s, but focused largely on the fluorogenic applications and did not attempt to develop or apply any labels of the pyrene sulfonic acids (cf., (i) Koller, E. and Wolfbeis, O., Continuous Kinetic Assay of Arylsulfatases with New Chromagenic and Fluorogenic Substrates, Analytica Chimica Acta 170, 73-80 (1985), and, (ii) Baustert, J. H., et al, Fluorometric Continuous Assay of α-Chymotrypsin Using new Protease Substrates Possessing Long Wavelength Excitation and Emission Maxima, Analytical Biochemistry 171, 393-397 (1988)). The sole exceptions to this focus on fluorogenic substrates in Koller's work were reported in 1989 in an article describing the lipophilic monoesters and diesters of monohydroxy pyrene trisulfonate and dihydroxypyrene disulfonate, respectively (Koller, E., Pyrene Sulfonates: An interesting class of fluorescent probes, Applied Fluorescence Technology 1, 13-14 (1989)).
In the early 1990s, Haugland and his colleagues (Whitaker, et al, Cascade Blue Derivatives: Water Soluble, Reactive Blue emission Dyes Evaluated as Fluorescent Labels and Tracers, Biochemistry 198, 119-130 (1991)) attempted to develop a panel of biological labels based upon water soluble derivatives of pyreneloxytrisulfonic acid which they designated “Cascade Blue” in recognition of the emission wavelengths of the
where R1 was chosen from the following panel of 20 esterified spacers comprising from 2 to approximately 15 carbons, with some spacers including dextran, t-BOC protecting groups, nitrogen, fluorine and oxygen as additional substituents:
1COCH32CO2H3CONH(CH2)2NH24CONH(CH2)6NH25CONH-DEXTRAN-NH26CONH(CH2)2NHCOCl7CONH(CH2)6NHCOCH2I8CO2-SUCC9CON310CONH(CH2)2OH11CONHNH212CONHNH213CONHNH2 14 15 16CONH(CH2)6CN 17 18 19CONH(CH2)6NHCOCH:CH220CONH(CH2)6NHCO(C6H6)N321CO2CH3 where R2 = OCH2CO2CH322CO2CH3 where R2 = —OH23CO2H where R2 = OCH2CO2H24CO2H where R2 = —OH
The general method of synthesis was condensation of an alkylating reagent such as an appropriately substituted alkyl halide, in the presence of a base, with a substituted or unsubstituted pyrene trisulfonic acid having a hydroxyl in the 1, 3, 6, or 8 position to give an alkoxy intermediate which was then hydrolyzed or reacted directly with hydrazine or aliphatic amines to give derivatives having the desired spacer and terminal functional group. Derivatives with terminal amines were further reacted with activated carboxylic acid derivatives, such as the succinimidyl esters of compounds possessing the desired additional reactive functional groups. In general, these Cascade Blue labels displayed 50 nanometer bandwidths, Stoke's Shifts valued between 20 and 30 nm, blue emission and relatively low molecular brightness, although quantum efficiencies were higher than with other blue emitting dyes (cf. Chemically reactive pyrenyloxy sulfonic acid dyes, U.S. Pat. No. 5,132,432 issued Jul. 21, 1992).